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Vol 9 No 6
Research article
The proinflammatory cytokines IL-2, IL-15 and IL-21 modulate the
repertoire of mature human natural killer cell receptors
Casimir de Rham
1
, Sylvie Ferrari-Lacraz
1
, Sabrina Jendly
1
, Gregory Schneiter
1
, Jean-Michel Dayer
2

and Jean Villard
1
1
Transplantation Immunology Unit, Department of Internal Medicine, University Hospital, rue Micheli-du-Crest, Geneva 14, 1211, Switzerland
2
Division of Immunology and Allergy, Department of Internal Medicine, University Hospital, rue Micheli-du-Crest, Geneva 14, 1211, Switzerland
Corresponding author: Jean Villard,
Received: 20 Jul 2007 Revisions requested: 27 Sep 2007 Revisions received: 23 Oct 2007 Accepted: 3 Dec 2007 Published: 3 Dec 2007
Arthritis Research & Therapy 2007, 9:R125 (doi:10.1186/ar2336)
This article is online at: />© 2007 de Rham et al.; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License ( />),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Natural killer (NK) cells play a crucial role in the immune
response to micro-organisms and tumours. Recent evidence
suggests that NK cells also regulate the adaptive T-cell
response and that it might be possible to exploit this ability to
eliminate autoreactive T cells in autoimmune disease and
alloreactive T cells in transplantation. Mature NK cells consist of
a highly diverse population of cells that expresses different
receptors to facilitate recognition of diseased cells and possibly
pathogens themselves. Ex vivo culture of NK cells with
cytokines such as IL-2 and IL-15 is an approach that permits
significant expansion of the NK cell subpopulations, which are
likely to have potent antitumour, antiviral, or immunomodulatory
effects in autoimmunity. Our data indicate that the addition of IL-
21 has a synergistic effect by increasing the numbers of NK
cells on a large scale. IL-2 and IL-15 may induce the expression
of killer cell immunoglobulin-like receptors (KIRs) in KIR-
negative populations, the c-lectin receptor NKG2D and the
natural cytotoxic receptor NKp44. The addition of IL-21 to IL-15
or IL-2 can modify the pattern of the KIR receptors and inhibit
NKp44 expression by reducing the expression of the adaptor
DAP-12. IL-21 also preserved the production of interferon-γ and
enhanced the cytotoxic properties of NK cells. Our findings
indicate that the proinflammatory cytokines IL-2, IL-15 and IL-21
can modify the peripheral repertoire of NK cells. These
properties may be used to endow subpopulations of NK cells
with specific phenotypes, which may be used in ex vivo cellular
immunotherapy strategies.
Introduction
Natural killer (NK) cells are an important population of lym-
phocytes that originally were regarded to play crucial roles in

protection from infectious disease and destroying tumour
cells; they are also involved in certain autoimmune diseases
and in rejection of transplanted tissues [1,2]. NK cells express
many different germline encoded activating or inhibitory recep-
tors that do not rearrange, in contrast to T and B cells, which
might suggest that NK cells are unable to respond to more
than a limited number of stimuli [3]. The human NK receptors
are characterized by genetic diversity, and NK cells were
found to express only a subset of these receptors [4,5]. NK
cells can be divided into CD56
bright
and CD56
dim
subpopula-
tions, the former being more inclined to produce cytokines
such as IFN-γ and the latter to lyse target cells [6]. Several acti-
vating and inhibitory NK cell receptors have been well charac-
terized, of which killer cell immunoglobulin-like receptors
(KIRs), c-type lectins, and natural cytotoxicity receptors
(NCRs). Although inhibitory receptors neutralize NK cells, acti-
vating receptors are responsible for NK cell activity [3,7-9].
The NK repertoire and its modulation at the cell surface is
incompletely understood. Many of the activating receptors are
constitutively expressed on all NK cells, and it is actually the
increased expression of their ligands on other cells induced by
mild stimuli that underpins the diversity of activating receptors.
The genetic diversity of NK cell receptors and the range of dis-
eases in which they are thought to play specific roles would
CFSE = 5-carboxyfluorescein diacetate succinimidyl ester; DAP-12 = DNAX-activating protein of 12 kDa; FACS = fluorescence-activated cell sort-
ing; FCS = foetal calf serum; IFN = interferon; IL = interleukin; KIR = killer cell immunoglobulin-like receptor; mAb = monoclonal antibody; NCR =

natural cytotoxicity receptor; NK = natural killer; NKG2D = NK group 2D; PBMC = peripheral blood mononuclear cell; PBS = phosphate-buffered
saline; PCR = polymerase chain reaction; rh = human recombinant.
Arthritis Research & Therapy Vol 9 No 6 de Rham et al.
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suggest that they might potentially be good therapeutic tar-
gets. To date, the suitability of KIR as a target of intervention
has been suggested by studies of bone marrow transplanta-
tion [10-12]. Exploitation of NK alloreactivity may become an
important therapeutic strategy in the management of myeloid
malignancy, in the modulation of engraftment procedures and
in the control of graft-versus-host-disease [13,14]. In autoim-
mune disease, NK cells can promote or inhibit the activation of
autoimmune T cells, and by virtue of their ability to rapidly kill
abnormal cells and produce cytokines and chemokines, NK
cells play a key role in regulating autoimmune responses.
Human studies and mice models suggest that the immu-
nomodulatory role of NK cells in autoimmunity is likely to pro-
vide new insights into the pathogenesis and treatment of
autoimmune disorders [15].
Because NK cells respond to cytokines, and because their kill-
ing activity can be enhanced by the presence of IL-2, some
investigators have suggested that adaptive transfer of NK cell
subsets in an activated state (after stimulation with IL-2, IL-12
and IL-15) may be necessary for optimal efficacy [16,17].
However, injection of proinflammatory cytokines such as IL-2
or IL-15 to activate endogenous NK cells may induce inflam-
mation and autoreactivity.
Therefore, expansion of NK cells ex vivo is a strategy that is
worth considering for several clinical applications. It remains to

be determined whether the increase in NK cells ex vivo after
exposure to cytokines modifies their peripheral repertoire. In
the mouse, cytokines such as IL-2, IL-15, IL-21 and IL-4 can
prompt considerable modifications and selective alterations in
the repertoire of murine NK cells [18]. In the present study we
assessed the effects of IL-2, IL-15 and IL-21 on the human NK
cell repertoire, and our findings indicate that addition of IL-21
to IL-2 or IL-15 induced a marked increase in NK cell numbers,
and that IL-2 and IL-15 may induce the expression of KIR
receptors in a KIR-negative fraction. Our data also indicate
that IL-21 can downregulate the expression of NKp44 recep-
tor via the adaptor DAP-12 while preserving production of
cytokines by the CD56
bright
and CD56
dim
subpopulations of
NK cells and even enhancing their cytotoxic function.
Materials and methods
Reagents and cytokines
RPMI-1640 medium, and β-mercaptoethanol were purchased
from Sigma Chemicals (St. Louis, MO, USA). Phosphate-buff-
ered saline (PBS), penicillin/streptomycin, L-glutamine, mini-
mal essential medium nonessential amino acids, and sodium
pyruvate were supplied by Gibco Invitrogen (San Diego, CA,
USA). Human AB serum was provided by the Blood Bank of
Geneva University Hospital (Geneva, Switzerland). Ficoll-
Paque™ Plus was from Amersham Biosciences (Uppsala,
Sweden). Human recombinant (rh)IL-2 was obtained from Bio-
gen Inc. (Cambridge, MA, USA), rhIL-15 was kindly provided

by Invitrogen (Seattle, WA, USA) and rhIL-21 was a gift from
Dr DC Foster (Zymogenetics, Seattle, WA, USA).
Isolation of NK cells, of CD56
dim
and CD56
bright
subpopulations, and cell cultures
Peripheral blood mononuclear cells (PBMCs) were isolated
from normal young donors by density-gradient centrifugation.
NK cells were separated from 300 × 10
6
PBMCs by negative
selection using an isolation kit (Miltenyibiotec, Bergisch Glad-
bach, Germany). Non-NK cells from human PBMCs, such as T
cells, B cells, dendritic cells, monocytes, granulocytes and
erythrocytes, were stained with a cocktail of biotin-conjugated
antibodies to CD3, CD4, CD14, CD15, CD19, CD36,
CD123 and CD123a. A second staining was conducted using
an antibiotin mAb conjugated with microbeads. The NK cells
were isolated by depletion of the magnetically labelled cells.
After negative selection, between 3% and 10% NK cells were
recovered (depending on the donor). NK cells were washed
with PBS and stained with APC-conjugated mAb to CD56
(Miltenyibiotec), biotin-conjugated mAb to CD16 (BD
Pharmingen™, San Diego, CA, USA) and APC-Cy7-conju-
gated mAb to CD3 (BD Pharmingen™). The unlabeled CD16
mAb was stained with streptavidin-ECD, a tandem dye com-
prising PE covalently linked to Texas-Red (BD Pharmingen™).
CD56
dim

and CD56
bright
NK cells were subsequently sepa-
rated on a FACSAria
®
sorter (BD Pharmingen™). The purity of
each subpopulation was consistently greater than 95%. The
selected NK cells were cultured at a concentration of 1 × 10
6
cells/ml for up to 7 days in RPMI medium supplemented with
the following: 10% HI AB serum, 100 U/ml penicillin, 100 μg/
ml streptomycin, 2 mmol/l L-glutamine, 1% minimal essential
medium nonessential amino acids and 0.1 mmol/l sodium
pyruvate, 5 mmol/l β-mercaptoethanol (at 5 × 10
-5
mol/l;
referred to as 'medium'). Then, rhIL-2 (25 ng/ml), rhIL-15 (25
ng/ml), rIhL-2 plus rhIL-21 (50 ng/ml), or rhIL-15 plus rhIL-21
was added. The NK cells were placed in 5% carbon dioxide-
air humidified atmosphere at 37°C.
Cytokine determination
Samples of conditioned medium were subjected to enzyme-
linked immunosorbent assay for determination of IFN-γ. The
sensitivity of all protein assays was 10 to 30 pg/ml. In addition
to enzyme-linked immunosorbent assay, an IFN-γ capture
assay kit (Miltenyibiotec) was used to determine the amount of
IFN-γ. After 7 days of culture with different cytokines, CD56
dim
and CD56
bright

NK cells were incubated for 45 min at 37°C
together with a bipolar anti-IFN-γ antibody, which binds to the
cells as well as to IFN-γ secreted on the cell surface. By using
a second antibody, namely PE-conjugated anti-IFN-γ, IFN-γ
was determined by fluorescence-activated cell sorting
(FACS).
Cell staining for flow cytometry
The following mouse anti-human mAbs were purchased from
BD Pharmingen™: PE-Cy7-conjugated anti-CD3; APC-Cy7-
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conjugated anti-CD16; FITC-conjugated anti-CD158b, which
recognizes KIR2DL2 (CD158b1), KIR2DL3 (CD158b2) and
KIR2DS2 (CD158j); PE-conjugated anti-CD158a specific for
KIR2DL1 (CD158a) and KIR2DS1 (CD158h); biotin-conju-
gated anti-NKB1 specific for KIR3DL1 (CD158e1); and APC-
conjugated anti-NKG2D. PE-conjugated anti-CD56, PE-con-
jugated anti-CD158i (KIR2DS4) and PE-conjugated anti-
NKG2A were supplied by Beckmann Coulter (Fullerton, CA,
USA). APC-conjugated anti-CD56, PE-conjugated anti-
NKp46, PE-conjugated anti-NKp44 and biotin-conjugated
anti-Nkp30 were from Miltenyibiotec, and PE-conjugated anti-
NKG2C was from R&D (R&D Systems Inc., Minneapolis, MN,
USA). The unlabeled NKB1 mAb was stained with streptavi-
din-ECD (Beckmann Coulter). For indirect immunofluores-
cence, nonspecific binding sites were saturated with normal
mouse serum before adding the relevant mAb. Six-colour
immunofluorescence was performed to assess surface marker
expression on CD56
dim

and CD56
bright
NK cells activated by
rhIL-2, rhIL-15, rhIL-2 plus rhIL-21, or rhIL-15 plus rhIL-21.
After 7 days of culture, CD56
dim
and CD56
bright
NK cells were
washed twice with PBS (completed with 2% foetal calf serum
[FCS]) and treated successively with FITC-, PE-, biotin-
streptavidin-ECD-, APC-, APC-Cy7-, and PE-Cy7-conjugated
mAbs on ice for 10 minutes and washed with PBS (completed
with 2% FCS). For the capture assay, NK cells were isolated
and washed once with PBS, complemented with 2% FCS.
IFN-γ was determined using the capture assay kit from Milteny-
ibiotec, in accordance with supplier's instructions. Cell stain-
ing was analysed using FACSAria
®
and FACS DIVA™
software (BD Pharmingen™).
CFSE labelling and analysis of NK cell proliferation in
vitro
A total of 400 × 10
6
human PBMCs were labelled with fluoro-
chrome 5-carboxyfluorescein diacetate succinimidyl ester
(CFSE; Molecular Probe, Inc., Portland, OR, USA), as
described previously [19]. CFSE was dissolved in dimethyl
sulfoxide and added to the cell suspension for 15 minutes at a

final concentration of 0.5 μmol/l at 37°C. The reaction was
stopped by the addition of PBS/10% FCS. The cells were
washed in PBS/10% FCS, resuspended in RPMI and left to
rest overnight at 37°C in a 5% CO
2
humid atmosphere. The
next day NK cells were isolated from 300 × 10
6
PBMCs
(stained with CFSE) using the NK cell isolation kit (see 'Isola-
tion of NK cells, of CD56
dim
and CD56
bright
subpopulations,
and cell cultures', above). Then, CD56
dim
and CD56
bright
NK
cells were separated on a FACSAria
®
sorter (BD Biosciences
PharMingen). The isolated CD56
dim
and CD56
bright
NK cells
were resuspended in RPMI and cultured at 1 × 10
6

cells/ml
with rhIL-2 (25 ng/ml), rhIL-15 (25 ng/ml), rhIL-2 plus rhIL-21
(50 ng/ml), or rhIL-15 plus rhIL-21 for 5 and 7 days. On days
5 and 7, cells were stained with several conjugated antibodies
(see 'Cell staining for flow cytometry', above) and six-colour
analysis by flow cytometry was performed on a Becton-Dickin-
son FACSAria
®
equipped with FACS DIVA™ software. Live
events were collected and analysed by gating on to CD56
dim
or CD56
bright
CFSE-positive cells.
Calculation of the frequency of proliferating NK cells
Proliferation of NK cells in response to cytokine stimulation
was analyzed as described previously [19]. By means of the
FACS acquisition software (FACS DIVA™), the total number of
cells in each generation of proliferation was calculated and the
number of precursors generating the daughter cells was
determined using the formula y/2n, where y is the number of
cells in each peak and n is the number of cell divisions. The fre-
quency of NK cell proliferation was then calculated by dividing
the total number of precursors by the total number of CFSE-
labelled cells.
Cytotoxicity assay
The isolated NK cells were cultured with rhIL-2 (25 ng/ml),
rhIL-15 (25 ng/ml), rhIL-2 plus rhIL-21 (50 ng/ml), or rhIL-15
plus rhIL-21 for 7 days. On day 7, NK cells were subjected to
the cytotoxicity assay. To test cytotoxicity, a standard

51
Cr-
release assay was performed. K562 cells were incubated for
1 hour with Na
2
51
CrO
4
(Hartmann Analytics, Braunschweig,
Germany), washed three times and co-incubated for 4 hours
with NK effector cells. The percentage of specific lysis was
calculated from the following formula: percentage of specific
lysis = ([experimental counts – spontaneous lysis]/[maximal
lysis – spontaneous lysis] × 100). Experiments were con-
ducted in triplicate.
RNA isolation and real-time PCR
Total cellular RNA was isolated from NK cells by lysing the
cells with Qiagen reagent and Qiagen Rneasy
®
Micro Kit (Qia-
gen AG, Basel, Switzerland), in accordance with the manufac-
turer's instructions. One microgram of RNA was treated with
DNase to eliminate any contaminating genomic DNA and sub-
sequently reverse transcribed. The quality of the reverse tran-
scription was tested for the expression of the housekeeping
gene 18S using real-time polymerase chain reaction (PCR).
Subsequently, the relative abundance of KIR genes was deter-
mined by TaqMan real-time PCR analysis on an ABI Prism
7300 Sequence detection instrument (Applied Biosystems,
Forster City, CA, USA). To quantify the levels of cDNA, the

expression of DAP12 and DAP10 was normalized against the
housekeeping gene 18S. Data were expressed as relative fold
difference between cDNA of the study samples (DAP12 at
day 7 with IL-15 and IL-15/IL-21) and a calibrated sample
(DAP12 at day 0). DAP12 (Hs00182426_m1), DAP10
(Hs00367159_m1) and 18S (4310893E) primer-probe sets
were purchased from Applied Biosystems (Foster City, CA,
USA).
Western blot analysis
Purified NK cells were cultured for 7 days with IL-15 (25 ng/
ml) or IL-15 plus IL-21 (50 ng/ml). On day 7, cells were har-
vested and resuspended at 4 × 10
6
cells/ml in 800 ml of ice-
Arthritis Research & Therapy Vol 9 No 6 de Rham et al.
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cold PBS and centrifuged. Total cell lysate was prepared and
subjected to Western blot analysis. The blots were probed
with anti-DAP12 (Santa Cruz Biotechnology, Santa Cruz, CA,
USA) and anti-β-tubulin (Sigma). A secondary horseradish
peroxidase-conjugated goat anti-rabbit (Dako, Glostrup, Den-
mark) was added for detection. Antibody-bound proteins were
detected by the Uptilight hrp Blot Chemiluminescence sub-
strate (Uptima, Montluçon, France).
Statistical analysis
Data were analyzed using two-factor analysis of variance test.
P < 0.05 and were considered significant (Statview 5.1 [SAS
Institute Inc., Cary, NC, USA] and GraphPad Prism 3.02
[GraphPad, Witzenhausen, Germany]).

Results
IL-21 acts in synergy with IL-2 or IL-15 to increase NK cell
subpopulations
To investigate the function of IL-21 in subpopulations of
human NK cells, we purified mature human NK cells by nega-
tive selection (Miltenyi beads; Figure 1a). CFSE-labelled pri-
mary NK cells and both CD56
bright
and CD56
dim
populations
were then cultured for up to 7 days in the presence of IL-2 or
IL-15 with or without the addition of IL-21 (Figure 1a,b). At
days 5 (Figure 1a [left panel]) and 7 (Figure 1a [right panel]),
addition of IL-21 to each IL-2 and IL-15 culture resulted in a
marked increase in cell division as compared with IL-2 and IL-
15 alone. As previously reported [20], IL-21 alone does not
induce NK cell proliferation (data not shown). In the next step,
CD56
bright
and CD56
dim
subpopulations were purified by
FACS to elucidate the role played by IL-21 in the expansion of
the NK cell subpopulations in the presence of IL-2 and IL-15
(Figure 1b). At day 7, IL-21 acted synergistically with IL-2 or
IL-15 in expanding the CD56
dim
population (Figure 1b [left
panel]). IL-15 alone proved sufficient to increase markedly the

CD56
bright
population, whereas IL-21 induced a significant
increase in the CD56
bright
population with IL-2 (Figure 1b [right
panel]). The increase in division mediated by IL-2 and IL-15
plus IL-21 was matched by an increase in total cell number of
NK cells. We observed a 10-fold increase in cell numbers with
IL-2/IL-21 or IL-15/IL-21 on the CD56
dim
population, as com-
pared with 3-fold and 2.8-fold increases with IL-2 and IL-15,
respectively. The expansion was more efficient on the
CD56
bright
population (an 18-fold and 28-fold increase with IL-
2 or IL-15 alone, as compared with a 40-fold and 50-fold
increase with IL-2/IL-21 and IL-15/IL-21, respectively; Figure
1c).
The KIR repertoire of NK subpopulations after seven
days of culture with IL-2 and IL-15 in the presence or
absence of IL-21
To analyze further the expression of the KIR repertoire on
CD56
bright
and CD56
dim
NK cell populations, we assessed the
expression of each KIR receptor on samples from five normal

donors before and after 7 days of culture with IL-2 and IL-15
alone or IL-2/IL-21 and IL-15/IL-21. Figure 2 and Table 1
depict a typical example of KIR 2DL1/S1 (anti-CD158a), KIR
2DL2/L3/S2 (anti-CD158b), KIR 3DL1 (anti-CD158e1) and
KIR 2DS4 (anti-CD158i) from a normal donor, detected at the
cell surface by multi-colour cytofluorometry. Because the anti-
bodies available failed to distinguish every activating and inhib-
itory KIR (with the exception of 2DS4 and 3DL1), or even
2DL2 and 2DL3, the KIR and KIR combinations recognized by
each antibody are discussed below. Of the CD56
dim
NK cell
population, 26% did not express any KIR at their cell surface
at day 0. 2DS4 was expressed by 37.5% of CD56
dim
NK cells
and only around 4% of the cells expressed at least one of the
other KIR combinations (2DL1/S1, 2DL2/L3/S2, or 3DL1). Of
CD56
dim
cells 20.21% expressed at least two KIR combina-
tions, mostly 2DS4 associated with 2DL2/L3/S2 (9.04%),
2DS4 with 2DL1/S1 (5.6%), and 2DS4 with 3DL1/S1
(2.24%) in this typical example. A very small fraction of
CD56
dim
expressed two KIR combinations without 2DS4
(0.80% to 1.64%). Three KIR combinations were expressed in
3.83% of CD56
dim

, mostly 2DS4 plus 2DL1/S1 plus 2DL2/
L3/S2 (2.32%), and only 0.17% expressed four KIR receptor
combinations at the cell surface. After 7 days of culture with
IL-2 or IL-15 and IL-2/IL-21 or IL-15/IL-21, the KIR repertoire
had not undergone any significant modification and had
remained virtually stable regardless of cytokine regimen, but
the percentage of KIR-negative cells tended to increase in the
presence of IL-21 (Figure 2a and Table 1).
As expected, a higher percentage of CD56
bright
NK cells failed
to express KIR at the cell surface (62.9%) [6]. Of CD56
bright
NK cells, 17.9% expressed 2DS4 only and around 7.86% of
CD56
bright
expressed at least one of the other KIR (2DL1/S1,
2DL2/L3/S2, or 3DL1) only. 2DS4 and one additional KIR
were expressed in 8.33% of the CD56
bright
cells. 2DS4 plus
2DL1/S1 plus 2DL2/L3/S2 were expressed in 2.38% of the
cells, and none of the other combinations of three or four KIR
was found at day 0. After 7 days of culture with the different
cytokines, the percentage of cells lacking KIR expression
increased with IL-21: 62.9% at day 0, 65% and 56% at day 7
with IL-2 and IL-15, respectively, versus 71.55% and 75.24%
at day 7 with IL-2/IL-21 and IL-15/IL-21, respectively. Expres-
sion by CD56
bright

cells of two or more KIRs fluctuated more
than that of CD56
dim
(Figure 2b and Table 1), but considering
the small percentages of these populations we have refrained
from drawing definitive conclusions from these results.
Several donors were tested, and each time between 25% to
45% of CD56
dim
cells failed to express any KIR, and 35% to
45% expressed predominantly one type of KIR (for example,
2DS4 in Figure 2). In CD56
bright
between 60% and 85% of the
cells did not express any KIR. We did not observe a selective
induction of KIR receptors at the cell surface of CD56
bright
and
CD56
dim
NK cells after 7 days of culture with IL-2 and IL-15
alone or IL-2/IL-21 and IL-15/IL-21, but the fraction of NK cells
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Figure 1
Ex vivo NK proliferation with IL-2 or IL-15 in the presence or absence of IL-21Ex vivo NK proliferation with IL-2 or IL-15 in the presence or absence of IL-21. (a) Proliferation of bulk natural killer (NK) cells. NK cells stained with
CFSE (5-carboxyfluorescein diacetate succinimidyl ester) were gated onto the CD3-negative fraction from peripheral blood mononuclear cells and
cultured for seven days with IL-2 or IL-15 in the presence or absence of IL-21. The number of cells undergoing division was analyzed at days 5 and
7. Proliferating NK cells are expressed in percentages. This experiment is representative of three individual experiments performed. (b) Proliferation
of NK cell subpopulations. NK cells stained with CFSE were purified by magnetic beads, and CD56

dim
and CD56
bright
subpopulations were isolated
by fluorescence-activated cell sorting. The two populations were cultured for 7 days with IL-2 or IL-15 in the presence or absence of IL-21 and ana-
lyzed at day 7. The percentage indicates proliferating NK cells. This experiment is representative of three individual experiments performed. (c)
Amplification of CD56
dim
and CD56
bright
subpopulations. Sorted 200,000 CD56
dim
and CD56
bright
NK cells were plated at day 0 and cultured for 7
days with IL-2 or IL-15 in the presence or absence of IL-21. At day 7 NK cells were stained for their purity (data not shown) and counted. The results
are expressed as fold increase as compared to day 0.
Arthritis Research & Therapy Vol 9 No 6 de Rham et al.
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without any KIR at their surface tended to increase in the pres-
ence of IL-21, especially on CD56
bright
NK cells.
IL-2 and IL-15 induce KIR expression on KIR negative
population
It is generally thought that the KIR receptors are acquired by a
stochastic mechanism, currently poorly understood, which
operates exclusively during NK cell development, and that the
repertoire is fixed after maturation. However, in mouse, Gays

and coworkers [18] showed that expression of the Ly49
receptor (the mouse equivalent of KIR in the human system
[18]) can be regulated by cytokines on mature NK cells.
To determine whether KIR would be expressed at the cell sur-
face of mature human NK cells, we focused more directly on
the KIR-negative population. We eliminated the KIR-positive
Figure 2
Expression of KIR repertoire after culture ex vivo of NK cells with IL-2 or IL-15 with/without IL-21Expression of KIR repertoire after culture ex vivo of NK cells with IL-2 or IL-15 with/without IL-21. (a) Expression of different killer cell immunoglobu-
lin-like receptor (KIRs) on the CD56
dim
natural killer (NK) population. NK cells were purified by magnetic beads, and the CD56
dim
subpopulations
were isolated by fluorescence-activated cell sorting (FACS) and cultured for seven days with IL-2 or IL-15 in the presence or absence of IL-21. Each
bar represents different culture conditions, and within the bar are shown the percentages of expression of KIR combinations: no KIR, and one, two,
three, or four KIR combinations. KIR combination signifies KIR recognized by a single antibody. Anti-CD158a recognizes KIR2DL1 (CD158a) and
KIR2DS1 (CD158h). Anti-CD158b recognizes KIR2DL2 (CD158b
1
), KIR2DL3 (CD158b
2
) and KIR2DS2 (CD158j). Anti-NKB1 is specific for
KIR3DL1 (CD158e
1
), and anti-KARp50.3 (CD158i) recognizes KIR2DS4. This experiment is representative of five individual experiments performed.
(b) Expression of the different KIR receptors on the CD56
bright
NK population. NK cells were purified by magnetic beads and the CD56
bright
subpop-
ulations were isolated by FACS sorting and cultured for 7 days with IL-2 or IL-15 in the presence or absence of IL-21. Each bar represents different

culture conditions, and within the bar are shown the percentages of expression KIR combinations: no KIR, and one, two, three, or four KIR combina-
tions. KIR combination signifies KIR recognized by a single antibody. Anti-CD158a recognizes KIR2DL1 (CD158a) and KIR2DS1 (CD158h). Anti-
CD158b recognizes KIR2DL2 (CD158b
1
), KIR2DL3 (CD158b
2
) and KIR2DS2 (CD158j). Anti-NKB1 is specific for KIR3DL1 (CD158e
1
), and anti-
KARp50.3 (CD158i) recognizes KIR2DS4. This experiment is representative of five individual experiments performed.
Available online />Page 7 of 15
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Table 1
The KIR phenotype on NK cells stimulated with IL-2, Il-15 and IL-21.
Day 0 Day 7 + IL-2 Day 7 + IL-15 Day 7 + IL-2/IL-21 Day 7 + IL-15/IL-21
CD56
dim
Negative 26.63 27.77 24.16 31.28 27.66
158i 37.57 38.81 40.33 36.53 40.00
158b1-b2-j 3.46 5.81 3.97 4.46 3.41
158e1 4.16 1.45 1.83 1.46 2.30
158a-h 3.97 2.71 3.22 3.23 3.27
158b1-b2-j/158i 9.04 12.29 11.28 11.57 8.92
158a-h/158i 5.60 3.77 5.37 3.76 4.71
158e1/158i 2.24 2.24 3.41 2.39 3.29
158a-h/158b1-b2-j 0.89 0.97 0.72 0.67 0.78
158e1/158b1-b2-j 1.64 0.35 0.72 0.61 0.66
158e1/158a-h 0.80 0.32 0.36 0.30 0.50
158a-h/158b1-b2-j/158i 2.32 2.06 2.02 1.75 2.06
158e1/158b1-b2-j/158i 0.68 0.63 1.31 0.96 1.30

158e1/158a-h/158i 0.42 0.50 0.66 0.50 0.70
158e1/158a-h/158b1-b2-j 0.41 0.10 0.22 0.21 0.14
158e1/158a-h/158b1-b2-j/158i 0.17 0.18 0.41 0.32 0.30
CD56
bright
Negative 62.86 65.69 56.57 71.55 75.24
158i 17.86 17.89 20.87 15.75 13.20
158b1-b2-j 1.67 6.11 8.34 5.45 5.48
158e1 2.38 1.87 2.17 1.29 1.24
158a-h 3.81 1.20 0.77 0.86 1.16
158b1-b2-j/158i 4.76 3.44 8.01 3.56 2.54
158a-h/158i 1.19 0.33 0.28 0.19 0.20
158e1/158i 2.38 0.62 0.51 0.22 0.36
158a-h/158b1-b2-j 0.71 1.00 0.23 0.15 0.12
158e1/158b1-b2-j 0.00 0.89 1.22 0.65 0.00
158e1/158a-h 0.00 0.13 0.10 0.00 0.16
158a-h/158b1-b2-j/158i 2.38 0.56 0.21 0.11 0.06
158e1/158b1-b2-j/158i 0.00 0.22 0.70 0.22 0.20
158e1/158a-h/158i 0.00 0.04 0.02 0.00 0.04
158e1/158a-h/158b1-b2-j 0.00 0.00 0.00 0.00 0.00
158e1/158a-h/158b1-b2-j/158i 0.00 0.00 0.00 0.00 0.00
Anti-CD158a recognize KIR2DL1 (CD158a) and KIR2DS1 (CD158h). Anti-CD158b recognize KIR2DL2 (CD158b1), KIR2DL3 (CD158b2) and
KIR2DS2 (CD158j). Anti-NKB1 is specific for KIR3DL1 (CD158e1). Anti-KARp50.3 recognize KIR2DS4 (CD158i). KIR, killer cell
immunoglobulin-like receptor.
Arthritis Research & Therapy Vol 9 No 6 de Rham et al.
Page 8 of 15
(page number not for citation purposes)
fraction by cell sorting and cultured the KIR negative fraction
in the presence of IL-2, IL-15, with or without IL-21. Figure
3a,b is representative of three separate experiments. Figure 3a

shows the KIR repertoire of a normal donor. In Figure 3b, we
show the purity of the KIR-negative population after the deple-
tion of the KIR-positive population by FACS sorting for the
same donor (left column). The following right column show the
expression of the KIR repertoire in a fraction of the KIR-nega-
tive cells after 7 days of culture with IL-2 and IL-15 with or
without IL-21. For every donor tested, 15% to 20% of the KIR-
negative fraction expressed KIR receptors at their cell surface
after 7 days of culture with IL-2 and IL-15 (Figure 3c). Interest-
ingly, the addition of IL-21 partially inhibited the expression of
KIR receptors (Figure 3b,c).
To rule out the possibility of a proliferation of a small residual
fraction of KIR-positive receptors, the NK cells were stained
with CFSE and the KIR-negative fraction was sorted by FAC-
SAria
®
. After 7 days of culture we noted that the NK cell pop-
ulation expressing the KIR receptor (Figure 4 [black
histogram]) underwent division to smaller extent than the KIR-
negative population (Figure 4 [grey histogram]), demonstrat-
ing that the KIR-positive NK cell population does not possess
a proliferative advantage. These results strongly suggest that
KIR receptors may be expressed de novo in the presence of
IL-2 or IL-15 in mature human NK cells.
NKp44 expression is induced by IL-2 and IL-15 and down-
regulated by IL-21
NCR receptors (NKp46, NKp44 and NKp30) are known to
mediate cytotoxicity to a variety of tumour target cells but also
to pathogen-specific antigens. Even though their ligands are
unknown, they appear to play a crucial role by activating NK

cells in the absence of additional stimuli [21]. NKp46 was
expressed by 100% of the CD56
bright
and CD56
dim
NK cell
populations and was not modified after 7 days of culture in the
presence of cytokines (Figure 5a,b [left]). NKp30 was also
expressed in 100% of the cells but to a lesser extent and it
was upregulated in both CD56
bright
and CD56
dim
NK cell pop-
ulations in the presence of IL-2, IL-15 and IL-21 (Figure 5a,b
[right]). Although NKp44 was not expressed by fresh primary
NK cells, it was expressed by both populations upon activation
by cytokines as previously described [22]. According to our
data, IL-2 and IL-15 induced NKp44 expression in 100% of
both NK populations (Figure 5a,b [centre]). The addition of IL-
21 to IL-2 or IL-15 downregulated NKp44 expression on
CD56
dim
and CD56
bright
NK cells (Figure 5a,b [centre]). Inter-
estingly, a fraction of CD56
bright
NK cells appeared to be
resistant to downregulation by IL-21 (Figure 5b [centre])

because 20% and 40% of cells treated with IL-2/IL-21 and IL-
15/IL-21, respectively, continued to express NKp44. The
NKp44 receptor mediates signal transduction through the
association of adaptor molecules, in particular DAP12 in
humans [23]. Given the importance of DAP12 in NKp44
expression, we conducted real-time PCR and Western blot to
analyze the expression of DAP12.
As shown in Figure 6a (left), the cell surface expression of
NKp44 was markedly reduced after 7 days of culture with IL-
15 and IL-21. In the same conditions DAP12 mRNA was
strongly reduced (Figure 6a [right]) and a 60% reduction in
DAP12 protein level after correction with the β-tubulin was
observed (Figure 6b). In the same experiment we confirmed by
FACS and real-time PCR that IL-21 downregulates NKG2D
and DAP10, respectively [24] (Figure 6c [left and right]).
These data suggest that IL-21 regulates both adaptors
DAP10 and DAP12, leading to reductions in expression of
NKG2D and NKp44.
NK cell subpopulations produce interferon-γ and
mediate cytotoxicity after proliferation ex vivo in the
presence of cytokines
Activation of NK cells in vivo is mediated by a variety of sig-
nals, leading to cytokine secretion and cytotoxic activity. After
the expansion ex vivo of NK cells in culture, it is crucial to
determine whether NK cells continue to kill target cells in
response to stimuli. Thus, NK cells were tested with respect to
their cytotoxicity to K562 target cells. Seven days of expansion
with the different cytokine regimens did not reduce the cyto-
toxicity to K562 target cells of NK cell population. The mean
cytotoxic activity of three experiments was higher with IL-15

and IL-21, as compared with IL-2 and IL-15 alone at a ratio of
10:1 (P = 0.02 and P = 0.04, respectively) and at the ratio of
1:1 (P = 0.015 and P = 0.05, respectively; Figure 7a).
Because IL-21 also downregulated NKp44/DAP12, we
wished to analyze the cytotoxicity of the NKp44-positive or -
negative faction. After day 7 of culture of purified NK cells with
IL-15 and IL-21, we separated the NKp44-positive fraction
from the NKp44-negative fraction and assessed their cytotox-
icity potential. Our data demonstrate that the NKp44/DAP12-
negative fraction is significantly more cytotoxic than the posi-
tive one at a ratio of 10:1 which was not fully expected [25].
Cytokines produced by activated antigen-presenting cells
such as IL-12 are potent stimulators of IFN-γ production by NK
cells. We therefore tested the IFN-γ production by CD56
dim
and CD56
bright
NK cells by IL-12 stimulation after 7 days of cul-
ture with IL-2 and IL-15, in addition to IL-21 (Figure 8a,b).
Although in the past the production of IFN-γ was considered
to be mostly confined to the CD56
bright
NK cell population [6],
our findings show that both populations responded substan-
tially and to similar extents to IL-12 stimulation in terms of IFN-
γ production. Moreover, without IL-12, a fraction of NK cells,
mostly in the CD56
dim
population, secreted IFN-γ after 7 days
of culture mainly in the presence of IL-21 (Figure 8a,b). The

production of IFN-γ detected by FACS capture assay was sub-
stantiated by enzyme-linked immunosorbent assay (data not
shown).
Available online />Page 9 of 15
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Discussion
In the present study, we optimized culture conditions to
enhance proliferation of mature human NK cells with IL-2 and
IL-15, in the presence or absence of IL-21, and we analyzed
the effect that addition of these cytokines had on the NK
receptor repertoire of the CD56
dim
and CD56
bright
subpopula-
tions of mature NK cells. The main results of this study are as
follows. First, IL-21 acts synergistically with IL-2 or IL-15,
enhancing markedly the CD56
dim
NK subpopulation. Second,
the KIR repertoire of NK cells was stable in culture, but the
KIR-negative cell fraction can express KIR receptors in culture
with IL-2 and IL-15, this production being less marked in the
presence of IL-21. Finally, IL-21, which is known to downreg-
ulate NKG2D, proved also to have the ability to downregulate
NKp44 (NCR receptor) induced by IL-15 and to enhance the
cytotoxicity of NK cells.
Several experimental studies have demonstrated the capacity
of NK cells to eliminate cancer cells. Evidence is now emerg-
ing that NK cells might also be a therapeutic target in autoim-

munity [2,26]. Two different strategies could be taken into
Figure 3
KIR expression in a population of KIR-negative NK cellsKIR expression in a population of KIR-negative NK cells. (a) Killer cell
immunoglobulin-like receptor (KIR) repertoire of a prototypical blood
donor at day 0. The KIR repertoire was assessed on the natural killer
(NK) bulk population at day 0 before the sorting of the KIR-negative
fraction. Anti-CD158a recognizes KIR2DL1 (CD158a) and KIR2DS1
(CD158h). Anti-CD158b recognizes KIR2DL2 (CD158b
1
), KIR2DL3
(CD158b
2
) and KIR2DS2 (CD158j). Anti-NKB1 is specific for
KIR3DL1 (CD158e
1
), and anti-KARp50.3 (CD158i) recognizes
KIR2DS4. This experiment is representative of three individual experi-
ments performed. (b) KIR repertoire of the KIR-negative population
after 7 days of culture. The KIR-negative population of the same donor
(panel a) was selected by fluorescence-activated cell sorting (FACS;
left column), and cultured for 7 days with IL-2 or IL-15 in the presence
or absence of IL-21 (the four columns to the right). The KIR repertoire
was assessed after 7 days of culture. Anti-CD158a recognizes
KIR2DL1 (CD158a) and KIR2DS1 (CD158h). Anti-CD158b recog-
nizes KIR2DL2 (CD158b
1
), KIR2DL3 (CD158b
2
) and KIR2DS2
(CD158j). Anti-NKB1 is specific for KIR3DL1 (CD158e

1
), and anti-
KARp50.3 (CD158i) recognizes KIR2DS4. This experiment is repre-
sentative of three individual experiments performed. The symbols used
are defined in panel a. (c) Effect of IL-2 and IL-15 and/or IL-21 on the
KIR repertoire of several donors. The KIR-negative population was
selected by FACS sorting (left column) and cultured for 7 days with IL-
2 or IL-15 in the presence or absence of IL-21. This experiment repre-
sents the fraction of KIR-negative sorted cells from five normal donors
who expressed KIRs after 7 days.
Figure 4
Proliferation of KIR-negative and KIR-positive NK cellsProliferation of KIR-negative and KIR-positive NK cells. Peripheral blood
mononuclear cells were stained with CFSE (5-carboxyfluorescein diac-
etate succinimidyl ester) and natural killer (NK) cells were purified by
magnetic beads. The CFSE-positive, killer cell immunoglobulin-like
receptor (KIR)-negative fraction was purified by fluorescence-activated
cell sorting before culture for 7 days with IL-2 or IL-15 in the presence
or absence of IL-21. The grey dots represent the KIR-negative fraction
and the black dots represent the KIR-positive fraction after 7 days of
culture (left panels). The percentage indicates the fraction KIR-positive
and KIR-negative NK cells after 7 days of culture. The number of cells
undergoing division in the KIR negative (in grey) and the KIR-positive (in
black) fraction were analyzed after 7 days of culture on CD56
+
/KIR
-
(in
grey) or CD56
+
/KIR

+
(black) gated cells (right panel).
Arthritis Research & Therapy Vol 9 No 6 de Rham et al.
Page 10 of 15
(page number not for citation purposes)
consideration: the activation of endogenous NK cells or their
expansion ex vivo. Taking into account the effects of IL-2, IL-
15 and IL-21 on the differentiation, maturation, proliferation
and activation of NK cells [9,27,28], these cytokines would
appear to be particularly suited for manipulating NK cells for
therapeutic purpose. Several clinical trials have helped to
assess the effect of IL-2 administration on activation and
expansion of endogenous NK cells [29,30]. Recent reports
have revealed the effect of IL-21 in preclinical models, sug-
gesting a strong antitumoural activity of IL-21 in renal cell car-
cinoma, melanoma and leukaemia [31]. However, all three of
IL-2, IL-15 and IL-21 have been implicated in autoimmunity,
and using such cytokines to activate endogenous NK cells
may favour inflammation and promote autoreactivity [9,27,28].
Ex vivo adaptive immunotherapy with NK cells has been tested
by several groups that have collected and purified clinical
grade NK cells before administering patients with doses of up
to 10
7
/kg, and IL-2 has already been used to increase num-
bers of NK cells in a therapeutic approach to melanoma, renal
carcinoma cells, or after haematopoietic stem cell transplanta-
tion [32-34]. However, it is supposed that small fractions of
NK cells characterized by specific phenotypes are responsible
for their antiviral, antitumoural, or immunomodulatory activity. In

addition, activation by different stimulus or manipulation of NK
cell subpopulations by genetic engineering could be much
efficient to design NK-cell based immunotherapeutic strate-
gies [35]. Therefore, starting off with a limited number of NK
cells would be of great interest for optimizing protocols for
increasing the number of NK cells ex vivo by preserving their
phenotypes.
Because of their intrinsic effects on NK cells, addition of IL-21
to IL-2 or IL-15 would be the best combination to optimize the
ex vivo proliferation of NK cells. Recent data demonstrated
that cultured NK cells survived better with IL-15 than with IL-2
in the presence of methylprednisolone, offering interesting
clues as to an appropriate NK cell cytokine conditioning regi-
men in adoptive immunotherapy [36]. In vitro, the effect of IL-
21 on the proliferation and differentiation of murine NK cells
proved insufficient to drive the proliferation of immature or
naïve NK cells; however, at low doses IL-21 enhanced a pro-
liferative response of these cells to either IL-2 or IL-15,
whereas high doses had an inhibitory effect [20]. Interestingly,
the number of functional NK cells in the peripheral lymphoid
organs of IL-21 receptor null mice and the number of bone
marrow NK cell precursors are similar in wild-type and γ c-defi-
cient mice, indicating that γ c-dependent cytokines are not
required for the earliest commitment events in the NK cell lin-
eage [37]. In addition, Gays and colleagues [18] demon-
strated that IL-21 and combinations of IL-21 and IL-15 or IL-4
can downregulate the expression of the NK gene complex
(NKC) and Ly49F receptors after maturation, resulting in an
enhanced lytic function. Consequently, in the mouse IL-21 is
not essential for NK cell development, but it may influence their

proliferation and their maturation into effectors cells.
In human, IL-21 was initially shown to stimulate the develop-
ment of NK cells in vitro [37] and is involved in the acquisition
of a mature KIR repertoire [38] from human bone marrow pro-
Figure 5
NCR repertoire expressed after culture ex vivo of NK cells with IL-2 or IL-15 with-without IL-21NCR repertoire expressed after culture ex vivo of NK cells with IL-2 or
IL-15 with-without IL-21. (a) Expression of the natural cytotoxicity
receptor (NCR) family on the CD56
dim
population. Natural killer (NK)
cells were purified by magnetic beads, and the CD56
dim
subpopulation
was isolated by fluorescence-activated cell sorting (FACS) and cul-
tured for 7 days with IL-2 or IL-15 in the presence or absence of IL-21.
NKp46, NKp44 and NKp30 were assessed on the CD56
dim
population
at days 0 and 7. One of three similar experiments is shown. (b) Expres-
sion of the NCR receptor family, NKp46, NKp44 and NKp30 on the
CD56
bright
population. NK cells were purified by magnetic beads and
the CD56
bright
subpopulations were isolated by FACS sorting and cul-
tured for 7 days with IL-2 or IL-15 in the presence or absence of IL-21.
NKp46, NKp44 and NKp30 were assessed on the CD56
bright
popula-

tion at days 0 and 7. One of three similar experiments is shown.
Available online />Page 11 of 15
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Figure 6
IL-21 downregulated cell surface expression of NKp44 and NKG2D and expression of DAP10 and DAP12IL-21 downregulated cell surface expression of NKp44 and NKG2D and expression of DAP10 and DAP12. (a) IL-21 downregulated NKp44 and
DNAX-activating protein of 12 kDa (DAP12). The percentage of natural killer (NK) cells expressing NKp44 at the cell surface after addition of IL-21
to IL-15 (left) was diminished. Simultaneously, transcripts of the adaptor DAP12 were quantified by real-time PCR after 7 days of culture with IL-15
and compared with the same culture conditions when IL-21 was added (right). The results indicate a reduction in DAP12 transcripts after addition of
IL-21 compared with day 0. The expression of DAP12 cDNA was normalized against the housekeeping gene 18S. Mean ± standard deviation of
three experiments is shown. (b) IL-21 downregulated DAP12 at the protein level. NK cells were purified by magnetic beads and cultured with IL-15
in presence or absence of IL-21. After 7 days of culture, the adaptor DAP12 protein was quantified by Western blot. The blots were probed with
anti-DAP12 and anti-β-tubulin antibodies. (c) IL-21 downregulated NKG2D and DAP10. The percentage of NK cells expressing NKG2D at the cell
surface after addition of IL-21 to IL-15 (left) is shown. Simultaneously, the adaptor DAP10 transcripts were quantified by real-time PCR after 7 days
of culture with IL-15 and compared with the same culture condition when IL-21 was added (right). As previously shown by Burgess and coworkers
[24], in the presence of IL-2, IL-21 down-regulated the expression of C-lectin receptor NKG2D at the cell surface and the transcript of the adaptor
DAP10 after 7 days of culture with IL-15. The expression of DAP10 cDNA was normalized against the housekeeping gene 18S. Mean ± standard
deviation of three experiments is shown.
Arthritis Research & Therapy Vol 9 No 6 de Rham et al.
Page 12 of 15
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genitors. IL-21 has also proven crucial to their adoption of a
fully functional cytotoxic capacity [31,37]. On human mature
NK cells, IL-21 was recently shown to downregulate NKG2D
on NK and T cells stimulated by IL-15 [39].
Like in the mouse [18], our results suggest that IL-2 and IL-15
induce similar effects in the human NK cells, both cytokines
being able to induce the expression of KIR receptors in a frac-
tion of KIR-negative populations. The addition of IL-21 pre-
vents KIR expression, at least to some extent. The paucity of
available KIR mAbs thus represents an obstacle to our study

and prevents us from drawing the formal conclusion that the
so-called KIR-negative NK cells do not express any activating
or inhibitory KIRs. It remains unclear why IL-2 and IL-15, with
or without IL-21, failed to modify significantly the KIR reper-
toire on the KIR-negative subpopulation analysed in NK bulk
populations (see Figure 2 and Table 1). The mechanisms
underlying the repertoire of a given NK population are not well
defined. NK cells do not express all their germline-encoded
receptors; instead, a selected combination of these receptors
is expressed in a stochastic manner [40]. In the mouse, the
pattern of expression of Ly49 receptors is determined by prob-
abilistic transcriptional switches in the promoter regions of the
genes and a similar mechanism may exist for KIR [41].
Cytokine environment and infection shape the NK repertoire,
which reflects a balance between activating and inhibitory
receptors. Signals delivered by cytokines through the JAK/
STAT (Janus kiase/signal transducer and activator of tran-
scription) transduction pathway modulate the expression of
the different receptors on NK cells to preserve an equilibrium,
maintaining self-tolerance and the capacity to produce
cytokines or to be cytotoxic. By placing a KIR-negative popu-
lation in culture, this equilibrium may be significantly modified,
resulting in the reactivation or resetting of KIR genes that in
turn are susceptible to cytokines.
We confirm recent data reported by Burgess and coworkers
[24]; those investigators reported that IL-21 inhibited DAP10
expression, leading to the downregulation of NKG2D [24]. In
addition, we show that IL-21 can inhibit expression of DAP12
(Figure 5). The fact that DAP12 also mediates NKp44 trans-
duction signalling but not that of NKp40 and NKp46 [21] may

account for the downregulation of NKp44 by IL-21. Inhibition
of DAP12 has several controversial effects. In the mouse
DAP12 knockout model, NK cells developed normally but the
activating Ly49 receptors were downregulated and nonfunc-
tional [42]. However, recent data also suggest that the
absence of DAP12 correlates with potent tumour rejection
mediated by NK cell activation [25]. Our results corroborate
these data [25], showing that the NKp44-negative population,
which has downregulated DAP12, is more cytotoxic than the
NKp44-positive population. Because DAP12 is also required
for the expression and signalling of KIR, mainly the KIR activa-
tor, we speculate that the reduction of KIR expression
observed after 7 days of culture with IL-21 shown in Figure
3b,c (right columns) could be due, at least in part, to the down-
regulation of DAP12.
In summary, our results strongly suggest that it is possible to
modify and ultimately even shape the repertoire of NK cells
receptors by modulating ex vivo mature NK cells by means of
cytokines. The addition of IL-21 to IL-15 or IL-2 increases sig-
nificantly the number of cells, with unaltered capacity to pro-
duce IFN-γ and even more potent cytotoxic activity, which is
quite similar to the effect of IL-21 on murine NK cell biology
Figure 7
Cytotoxicity of NK cells after ex vivo proliferation in the presence of cytokinesCytotoxicity of NK cells after ex vivo proliferation in the presence of
cytokines. (a) Cytotoxicity of natural killer (NK) cells to K562 cells.
Human primary NK cells were purified by magnetic beads and cultured
for 7 days with IL-2 or IL-15 in the presence or absence of IL-21. Both
populations were analyzed for cytoxicity to K562 target cells at ratios of
10:1 and 1:1. One of three similar experiments is shown. (b) Cytotoxic-
ity of NKp44-positive and NKp44-negative NK cells. Human primary NK

cells were purified by magnetic beads (bulk) and cultured for 7 days
with IL-15 and IL-21. The NKp44-positive and NKp44-negative sub-
populations were sorted by fluorescence-activated cell sorting and
were analyzed for their cytotoxicity to K562 targets cells at ratios of
10:1 and 1:1. One of three similar experiments is shown.
Available online />Page 13 of 15
(page number not for citation purposes)
Figure 8
Expression of IFN-γ on NK cells after their proliferation ex vivo in presence of cytokinesExpression of IFN-γ on NK cells after their proliferation ex vivo in presence of cytokines. IFN-γ was analyzed by capture assay on natural killer (NK)
cells at day 0 without cytokines, and after 7 days of culture with IL-2 or IL-15 in the presence or absence of IL-21. To determine the capacity of NK
cells to produce IFN-γ, cells were subjected to stimulation by IL-12 for 12 hours under all conditions. One of three similar experiments is shown.
+

+ IL-12
B. CD56
bright
A. CD56
dim
+

+ IL-12
+IL-15/
IL-21
+IL-2/
IL-21
+IL-15
+IL-2
CD56
IFN-
J

D0
D7
Arthritis Research & Therapy Vol 9 No 6 de Rham et al.
Page 14 of 15
(page number not for citation purposes)
[18,41]. With regard to immunotherapy, this insight is funda-
mental because with this ex vivo strategy it may become pos-
sible to envisage tailoring specific phenotypes to
subpopulations of NK cell. Human cancer patients would cer-
tainly be the first to benefit from a strategy consisting of NK
cell based immunotherapy, but one may also envisage, in the
near future, treatment of autoimmune diseases after amplifying
ex vivo NK cell populations possessing regulatory properties
[43].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CdR designed, performed, analyzed and interpreted experi-
ments and wrote the paper. SFL designed, performed, ana-
lyzed and interpreted experiments, contributed to protocol
design and wrote the paper. SJ and GS performed experi-
ments. JMD was responsible for intellectual and financial con-
tributions. JV designed and interpreted experiments, wrote the
paper, designed the protocol, and oversaw all aspects of the
experiments.
Acknowledgements
We are indebted to Dr DC Foster (Zymogenetics, Seattle, WA, USA) for
providing human recombinant IL-21 and to Invitrogen for providing
human recombinant IL-15.
We are also grateful to Roswitha Rehm for critical reading of the manu-

script. This work was supported by Geneva University Hospital, the
Department of Internal Medicine, the Dubois-Ferrière-Dinu-Lipatti Foun-
dation (to JV), and the Swiss National Science Foundation, grants Nos
310000-108453 and PMPDA-110347 (to SFL).
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